Expression and diagnostic use of gag encoded peptides which are immunologically reactive with antibodies to HIV

ABSTRACT

Immunologically reactive gag proteins of LAV are expresesed in bacterial cells. The gag proteins are encoded by a recombinant plasmid containing procaryotic transcriptional and translational signals for expression, followed downstream by a DNA sequence comprising a portion of the gag region of LAV. Preferred signals for expression are selected from an inducible and/or suppressible operon, such as the trp operon. The gag proteins are isolated from the bacterial host and are utilized in diagonstic assays which detect the presence of LAV antigens or antibodies immunologically reactive with LAV. Further, the proteins produced by the method disclosed may be used as a vaccine against infection by the caustive virus for acquired immune deficiency syndrome.

This is a continuation of application Ser. No. 06/763,460, filed Aug. 7.1985, now abandoned.

TECHNICAL FIELD

The present invention relates generally to the expression of viralproteins through the use of recombinant DNA technology, and morespecifically, to the expression of proteins which are immunologicallyreactive with antibodies to lymphadenopathy-associated virus (LAV), nowknown as Human Immunodeficiency Virus (HIV).

BACKGROUND ART

Acquired immune deficiency syndrome (AIDS) is a transmissible deficiencyof cellular immunity characterized by opportunistic infections andcertain rare malignancies. The dominant risk groups for AIDS includehomosexually active males, intravenous drug abusers, recipients oftransfusions and blood products, and the heterosexual partners andchildren of high-risk individuals, suggesting the involvement of aninfectious agent transmitted through intimate contact or blood products.

Recent evidence indicates that the infectious agent responsible fordisease transmission is a novel lymphotropic retrovirus, known aslymphadenopathy-associated virus (LAV) (Barre-Sinoussi et al., Science220: 868 (1983)). Similar viruses have been reported by other scientificgroups (Popovic et al., Science 224: 497 (1984); Levy et al., Science225: 840 (1984); Vilmer et al., Lancet 1:753 (1983)) and designatedhuman T-cell lymphotropic virus type III (HTLV-III), AIDS-associatedretrovirus (ARV), or immune deficiency-associated virus (IDAV). Stillmore recent data indicates that LAV, HTLV-III, ARV, and IDAV shareseveral important characteristics, including substantial nucleotidehomology (Wain-Hobson et al., Cell 40:9 (1985); Muesing et al., Nature313: 450 (1985); Sanchez-Pescador et al., Science 227: 484 (1985)), andshould be considered isolates of the same virus, although there is alikelihood that strain-to-strain variations among the viral isolateswill exist. In addition to exhibiting substantial nucleotide homology,the isolates are similar with respect to morphology, cytopathology,requirements for optimum reverse transcriptase activity, and at leastsome antigenic properties (Levy, supra; Schupbach et al., Science 224:503 (1984)).

As noted above, the virus is known to be transmissible through bloodproducts (blood, blood serum, blood plasma, and fractions thereof),making it important to screen the blood products to determine if thedonor has been exposed to the virus. This can be done in any of severalways, including enzyme-linked immunosorbent assay (ELISA) for thedetection of antibodies to LAV and related viruses. Individuals whoseblood contains antibodies to LAV are said to be "seropositive." Bloodfrom seropositive donors may be eliminated from the blood supply upondetection, thereby helping to prevent the spread of the disease.

The immune response of individuals exposed to LAV is variable.Antibodies can be produced to any of several viral proteins, includingp13, p18, p25, p36, gp43, p55, gp110, etc. (Schupbach et al., N. Engl.J. Med. 312: 265 (1985)). Not all individuals will make antibodies tothe same proteins or to the same epitope on a given protein.

The detection of seropositive individuals, as currently practiced, hasseveral inherent problems. Foremost among these problems is the need toisolate antigen from whole viruses for the immunological assays. Thisisolation requires the manipulation of large volumes of live,potentially infectious virus, and as such poses a significant safetyhazard. In addition, there are concerns relating to the yield, purity,and reproducibility of virus from one preparation to another. This mayresult in an unacceptable number of false positives and/or negatives.Consequently, there is a need in the art for alternative methods ofproducing viral antigens which are useful in blood screening assays, andwhich further provide other related advantages.

DISCLOSURE OF INVENTION

Briefly stated, the present invention discloses DNA sequences comprisinga portion of the group specific antigen (gag) region of the LAV genome,the portion coding for a protein which is immunologically reactive withantibodies to LAV. A recombinant plasmid capable of replication inbacterial host cells is also disclosed. The plasmid includes procaryotictranscriptional and translational signals for expression, followed inreading phase by the DNA sequence described above. In a preferredembodiment, signals are chosen from an operon, such as the trp operon,which is inducible and/or suppressible. Bacterial cells, such as E.coli, which have been transformed with the recombinant plasmid describedabove, are also disclosed.

Another aspect of the invention discloses a method for preparingproteins which are immunologically reactive with antibodies to LAV. Themethod comprises introducing into a bacterial host cell a recombinantplasmid capable of replication in bacterial host cells. The plasmidincludes procaryotic transcriptional and translational signals forexpression, followed in reading phase by a DNA sequence comprising aportion of the gag region of the LAV genome, the portion coding for aprotein which is immunologically reactive with antibodies to LAV.Subsequent to the introduction of the plasmid, the bacterial host isgrown in an appropriate medium. Expression of the protein is theninduced and the protein product of the sequence is isolated from thebacterial host. The protein product may be purified subsequent toisolation, as by gel permeation chromatography.

A further aspect of the invention discloses a method for determining thepresence of antibodies to LAV in a biological fluid. The methodcomprises incubating the biological fluid with a protein produced bybacterial cells transformed with a recombinant plasmid as describedabove, thereby forming a reaction mixture, and subsequently analyzingthe reaction mixture to determine the presence of the antibodies. In apreferred embodiment, the step of analyzing the reaction mixturecomprises contacting the reaction mixture with a labeled specificbinding partner for the antibody.

Yet another aspect of the invention discloses a method for determiningthe presence of LAV antigen in a biological fluid, comprising incubatingthe biological fluid with a labeled protein produced by bacterial cellstransformed with a recombinant plasmid as described above, and eithersequentially or simultaneously, with an antibody to the protein suchthat specific binding occurs. Subsequently, the reaction mixture formedduring the incubation is analyzed to determine the amount of labelassociated with the antibody.

A method for producing antibodies to LAV comprising immunizing an animalwith a protein produced by bacterial cells transformed with arecombinant plasmid as described above, is also disclosed.

An additional aspect of the present invention discloses a method fordetermining the presence of antibodies to LAV in a biological fluid,comprising conjugating latex beads to a protein produced by bacterialcells transformed with a recombinant plasmid capable of replication inbacterial host cells, the plasmid including procaryotic transcriptionaland translational signals for expression. The signals are followed by aDNA sequence comprising a portion of the gag region of the LAV genome,the portion coding for a protein which is immunologically reactive withantibodies to LAV. Subsequently, the biological fluid is incubated withthe bead/protein conjugate, thereby forming a reaction mixture. Thereaction mixture is then analyzed to determine the presence of theantibodies.

The proteins produced within the present invention, when used with asuitable carrier or diluent, form an immunologically effective vaccinecomposition. By administering to an individual an immunogenicallyeffective amount of a protein encoded by a DNA sequence comprising aportion of the gag region of the LAV genome attached to aphysiologically acceptable carrier, infection caused by the virusresponsible for AIDS can be prevented.

Other aspects of the invention will become evident upon reference to thefollowing detailed description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of pSS-5 and pBS-5 from λJ19.

FIG. 2 illustrates the trp E expression vectors pJH 12 and pJH 14,including the polylinker sequences.

FIG. 3 illustrates the origin of the LAV inserts in pGAG-2 and pGAG-3.

FIG. 4 illustrates the construction of pGAG-2 from pJH 12 and pSM002.

FIG. 5 illustrates the construction of pGAG-3 from pJH 14 and pBPB14.

BEST MODE FOR CARRYING OUT THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

Lymphadenopathy-Associated Virus (LAV): A human T-lymphotropicretrovirus. For purposes of the present invention, a virus is consideredto be the same as or equivalent to LAV if it substantially fulfills thefollowing criteria:

(a) the virus is tropic for T-lymphocytes, especially T-helper cells(CD4⁺, according to the international nomenclature defined in Bernard etal., eds., Leucocyte Typing,New York: Springer Verlag (1984));

(b) the virus is cytopathic for infected CD4⁺ cells (rather thantransforming, as are HTLV-I and II);

(c) the virus encodes an RNA-dependent DNA polymerase (reversetranscriptase) which is Mg²⁺ -dependent (optimum concentration 5 mM,optimum pH 7.8, not inhibitable by actinomycin D) and can employ oligo(dT)₁₂₋₁₈ as a primer for reverse transcription from its 3' LTR;

(d) the virus bands in a sucrose gradient at a density of approximately1.16;

(e) the virus can be labeled with [³ H] uridine;

(f) the virus is distinct by immunological and nucleotide sequencecriteria from members of the HTLV-I/II family of viruses (by thiscriterion HTLV-III is not to be considered a member of the HTLV-I/IIfamily);

(g) the virus is substantially cross-reactive immunologically with theproteins encoded by the gag and env regions of LAV; and

(h) the virus shares substantial nucleotide homology (75-100%) and aminoacid sequence homology (75-100%) with LAV.

Immunologically Reactive: An antigen and an antibody are said to be"immunologically reactive" if they are capable of binding specificallyto each other, typically with an affinity of at least 10⁶ M⁻¹, moreoften at least 10⁸ M⁻¹.

Transformed or Transformation: The process of stably and heritablyaltering the genotype of a recipient cell or microorganism by theintroduction of purified DNA.

Lymphadenopathy-associated virus (LAV) can be isolated from patientswith AIDS or lymphadenopathy syndrome. The lymph nodes of such patientsare typically biopsied and placed in culture medium supplemented asnecessary to support growth. A mitogen such as interleukin-2 (IL-2) orphytohemagglutinin (PHA) can be included; antiserum to human interferoncan also be included. Reverse transcriptase activity typically appearsabout day 15 of culture, indicating the presence of virus. The virus canbe concentrated from the culture supernatant using a non-ionicdetergent, followed by banding in a sucrose gradient. These and othermethods of purification are well known in the art and are described, forexample, in Montelaro et al., J. Virology 42: 1029 (1982).

LAV can be propagated in any of a number of ways. It can be cultured inT-lymphocytes derived from umbilical cord or peripheral blood or frombone marrow. Alternatively, it can be propagated in immortalized T-cellsor B-cells; see, for example, Popovic et al., Science 224: 497 (1984),and Montagnier et al., Science 225: 63 (1984). Growth of the virus isusually monitored by the presence of reverse transcriptase activity.

A genomic clone of LAV can be prepared by any of several methods wellknown in the art, including but not limited to those described by Hahnet al., Nature 312: 166 (1984); Alizon et al., Nature 312: 757 (1984);Luciw et al., Nature 312: 760 (1984); and Muesing et al., Nature 313:450 (1985).

Briefly, in one of these methods (Alizon et al.) DNA is isolated fromLAV-infected T-cells of a healthy donor, partially digested with arestriction endonuclease such as Hind III, and the resultant digestfractionated electrophoretically. Fragments which correspond in size tothe size of the entire LAV genome (approximately 9.2 Kb) are eluted fromthe gel, precipitated, resuspended, and ligated into the arms of anappropriately restricted vector. The ligation mix is packaged intobacteriophage particles. Bacteria are transformed with thebacteriophage, and the clones are screened in situ for LAV inserts usinga suitable probe (such as cDNA made from LAV-RNA). From a positiveclone, the desired region of LAV can be subcloned into a bacterialplasmid vector, such as pUC 18. Further subcloning can be desirable toremove unwanted sequences and to add additional restriction sites (inthe form of a polylinker) at either end for the purpose of facilitatingcloning into an expression vector.

The LAV sequences can then be subcloned into an inducible expressionvector. A variety of expression vectors are known in the art and includeλ gt 11:Tn5 (Hall et al., Nature 311: 379 (1984); trp E (Paul et al.,Eur. J. Cell Biol. 31: 171 (1983); pINIII (Masui et al., Biotechnology,Jan. 1984, p. 81).

The resultant proteins can be partially purified and used for a varietyof purposes, including, as immunogens and antigens in immunoassays. Foruse as immunogens, the proteins can be injected into an animal, such asa mouse, rabbit, goat, etc., either in buffered solution or in adjuvant.Alternatively, the proteins can be purified by polyacrylamide gelelectrophoresis and the bands of interest excised from the gel,triturated, and resuspended in buffer for injection into the hostanimal. Polyclonal or monoclonal antibodies can be prepared. For use asantigens in immunoassays, the proteins can be employed in labeled orunlabeled form. Where they are labeled, the labels can includeradioisotopes, fluorophores, enzymes, luminescers, or particles. Theseand other labels are well known in the art and are described, forexample, in the following U.S. Pat. Nos. 3,766,162; 3,791,932;3,817,837; 3,996,345; and 4,233,402.

Assays employing the recombinant proteins of the instant invention canbe heterogeneous (i.e., requiring a separation step) or homogeneous. Ifthe assay is heterogeneous, a variety of separation means can beemployed, including centrifugation, filtration, chromatography, ormagnetism.

One preferred assay for the screening of blood products or otherphysiological fluids for the presence of antibodies is an ELISA.Typically, antigen (in this case, one or a combination of recombinantproteins) is adsorbed to the surface of a microtiter well. Residualprotein-binding sites on the surface are then blocked with anappropriate agent, such as bovine serum albumin (BSA), heat-inactivatednormal goat serum (NGS), or BLOTTO (a buffered solution of nonfat drymilk which also contains a preservative, salts, and an antifoamingagent). The well is then incubated with a sample suspected of containingspecific antibody. The sample can be applied neat, or, more often, itcan be diluted, usually in a buffered solution which contains a smallamount (0.1-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO.After incubating for a sufficient length of time to allow specificbinding to occur, the well is washed to remove unbound protein and thenincubated with labeled anti-human immunoglobulin (αHuIg). The label canbe chosen from a variety of enzymes, including horseradish peroxidase(HRP), β-galactosidase, alkaline phosphatase, and glucose oxidase.Sufficient time is allowed for specific binding to occur, then the wellis again washed to remove unbound conjugate, and the substrate for theenzyme is added. Color is allowed to develop and the optical density ofthe contents of the well is determined visually or instrumentally.

For convenience, reagents for ELISA may be titer plates to which viralproteins made by recombinant techniques have been pre-adsorbed, variousdiluents and buffers, labeled conjugates for the detection ofspecifically bound antibodies, and other signal-generating reagents,such as enzyme substrates, cofactors, and chromogens.

Sera of LAV-infected individuals contain antibodies to many LAVproteins, including p13, p18, p25, p36, gp43, p55, gp110, etc. Althoughnot all individuals make antibodies to the same proteins, individualsera are most consistently reactive with antibodies to the gag proteins,p25 and p18, and to the env proteins, gp43 and gp110. Variation betweenindividuals may be due to several factors, including diseaseprogression. For example, there is some evidence that antibodies to coreproteins are prominent during the earliest stages of the disease, butdecline with progression of immune suppression. In contrast, antibodytiters to the envelope glycoproteins are thought to persist during thelater stages of the disease.

Additional variation in response may be due to polymorphism in the genesencoding viral proteins. Different isolates of LAV possess significantalterations in the env protein. Interestingly, the gag protein sequencesare highly conserved.

In every seropositive sample examined, antibodies to at least one of thegag proteins (p18 or p25) or one of the env proteins (gp43 or gp110)have been seen. However, none of these proteins are universallyrecognized by seropositive individuals. It therefore seems essentialthat blood be screened for antibodies to at least one gag and one envprotein. In a previous patent application, U.S. Ser. No. 721,237,entitled "Expression of Immunologically Reactive Viral Proteins," thedisclosed invention utilizes portions of the envelope (env) region ofthe LAV genome, which codes for a protein which is immunologicallyreactive with antibodies to LAV. The present invention utilizes portionsof the gag region of the LAV genome, which codes for a protein which isalso immunologically reactive with antibodies to LAV. In combination,proteins encoded by the gag and env regions can be utilized to detectseropositive individuals with a high degree of sensitivity.

Another application of the recombinant proteins of this invention is asvaccines for human use. The recombinant proteins can be extensivelypurified and formulated in a convenient manner, generally inconcentrations of 1 ug to 20 mg per kg of host. Physiologicallyacceptable carriers, such as sterile water, saline, buffered saline,etc., can be employed. Adjuvants, such as aluminum hydroxide, can alsobe employed. The vaccine can be administered by intravenous,subcutaneous, intramuscular, or peritoneal injection. One injection canbe sufficient, but more often, multiple injections at weekly to monthlyintervals are preferred.

Alternatively, vaccinia virus recombinants can be constructed whichexpress regions of the LAV genome. For example, the constructs of thisinvention can be inserted into a plasmid such as pMM34 (Mackett et al.,Science 227: 433, 1985) and vaccinia virus hybrids containing theresultant chimeric plasmid, formed by homologous recombination.Immunization with such recombinant virus vaccines has been shown to beeffective in eliciting protective immunity in animals to hepatitis Bvirus and vesicular stomatitis virus (Smith et al., Nature 311: 578,1984).

The use of a recombinant protein vaccine in this manner eliminates theneed to compose vaccines from inactivated preparations or a virulentstrains of pathogenic microorganisms.

In the following example, two overlapping regions of LAV-gag wereselected for expression (FIG. 3). The choice was influenced by thefinding that p18 and p25 were the gag proteins most reproduciblyreactive with sera from LAV-infected individuals. Our selection withinthese sequences was dictated by the location of hydrophilic regions andprotease cleavage sites (both of which may be exposed at the surface ofthe protein and immunogenic) as well as by the size limitation forefficient expression in the chosen vectors (trp E).

The LAV genomic clone designated λ J19 was subcloned into the bacterialplasmid vector, pUC 18. The resultant subclone, designated pBT-1, wasfurther subcloned to yield pSS-5 and pBS-5, which containedpredominantly gag and pol sequences. The gag sequences were furthersubcloned into pIC19R, (forming plasmids pSM002 and pBPB14), and thenregions of the gag sequence were transferred into the trp E inducibleexpression vector. The gag DNA was inserted in-frame downstream of thetrp E gene, resulting in the expression of a trp E-gag fusion proteinwhen E. coli were transformed with this construct. The resultantproteins were partially purified and characterized by their reactivityin ELISA with sera from known seropositive and known seronegativeindividuals. Two useful constructions, designated pGAG-2 and pGAG-3,were identified.

The following example is offered by way of illustration, and not by wayof limitation.

EXAMPLE

A. Construction of the trp-gag expression vectors

Any of several bacterial expression systems can be used to expressforeign proteins. The trp E system was chosen for the expression ofLAV-gag sequences because it contains a strong inducible promoter, butits expression can also be suppressed so that foreign (and potentiallytoxic) protein does not accumulate within the bacteria for long periodsof time.

Expression vectors are limited by the type and reading frame of theirrestriction sites. (or example, the trp E expression vectors requirethat the DNA insert possess Bam HI, Hind III, or EcoRI restriction sitecompatible termini. More diversity can be introduced by first subcloningthe region of interest into an intermediate vector which possesses abroader range and altered arrangement of restriction sites. The regionof interest can then be introduced into an expression vector by usingrestriction sites provided by the intermediate vector.

Our strategy, therefore, was to first subclone most of the gag region ofthe LAV genome into a transfer vector, pIC19R. Then, various fragmentsof these subclones were ligated into either of two trp expressionvectors (pJH 12, pJH 14), which differed only in the reading frame ofthe restriction sites in the polylinker region (see FIG. 2).

1. Subcloning LAV genome

a. Preparation of phage DNA

The entire LAV genome was obtained from the Pasteur Institut in the formof λ phage particles containing a 9.2 Kb genomic DNA insert in the HindIII site of phage λ L47.1. This clone is referred to as λ J19 and isdescribed in Wain-Hobson et al., Cell 40: 9 (1985). λ J19 phageparticles were transfected into the Q359 strain of E. coli K-12 (thegenotype of Q359 is hsdRk⁻, hsdMk⁺, supF, φ80, P2) according to theprocedure of Maniatis et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Laboratory, 1982, at p. 64. A single plaquewas picked and the phage amplified by the plate lysate method (Maniatis,supra, at p. 65). After a nine-hour incubation at 37° C., the plates(100 mm diameter) containing confluent plaques were overlaid with 5 mlof 100 mM NaCl/20 mM MgSO.sub. 4 /50 mM Tris, pH 7.5. After incubatingfor twelve hours at 4° C., the liquid was collected and extracted twotimes with an equal volume of chloroform.

To 10 ml of the resultant aqueous phase containing phage particles wasadded 2 ml 0.25 M EDTA/2.5% SDS/0.5 M Tris, pH 9, and the suspension wasincubated at 70° C. for fifteen minutes to disrupt the phage. 2.5 ml 8 Mpotassium acetate was added, and the solution was incubated on ice forfifteen minutes, then centrifuged for ten minutes at 12,000 xg at 4° C.to pellet protein. The supernatant was transferred to a 50 mlpolypropylene centrifuge tube and extracted with an equal volume ofphenol (pH 8, equilibrated with 1 M Tris, pH 8) at 20° C. The aqueousphase was then extracted with an equal volume of chloroform:isoamylalcohol (24:1) at 20° C. To the aqueous phase was then added 2.5 volumesof 95% ethanol to precipitate the DNA. After centrifugation, the DNApellet was dried and resuspended in 10 mM Tris HCl, pH 7.4/1 mM EDTA.

b. Subcloning the gag region

Approximately 12 ug of λ J19 DNA prepared in A.l.a above was digested tocompletion with the restriction enzyme Sst I (Bethesda Research Labs,Bethesda, MD), which only cuts in the LTR regions of this isolate ofLAV. The digest mixture was electrophoresed at 1 V/cm through 0.9%agarose in 0.089 M Tris-borate/0.089 M boric acid/1 mM EDTA. Theposition of the 9.1 Kb fragment was determined relative to molecularweight standards after staining with ethidium bromide. This band waselectroeluted into NA45 paper (Schleicher and Schuell, Keene, NH). TheDNA was recovered from the paper according to instructions provided bythe manufacturer.

The 9.1 Kb Sst I fragment was ligated into the Sst I digested vector pUC18, at a ratio of 10 insert molecules: 1 vector molecule. E. coli strainHB101 was transformed with the ligation mix by the CaCl₂ procedure ofManiatis et al. (supra) and plated onto LB plus ampicillin (200 ug/ml)agar plates.

Single colonies were picked and diluted into 3 ml LB plus ampicillinmedium and grown overnight at 37° C. with constant shaking. Plasmid DNAwas prepared by the alkaline lysis method (Maniatis et al., supra, at p.368). One colony was selected which contained the 9.1 Kb Sst I insert inan orientation such that the Eco RI site in the polylinker was closestto the 5' end of the LAV genome, as determined by restriction analysisof the plasmid DNA. This subclone was designated pBT-1 (ATCC Accession#53069) (FIG. 1).

Two subclones were made from plasmid pBT-1 (see FIG. 1). For the first(pSS-5), pBT-1 was digested with Sal I and then religated. For thesecond, pBS-5, pBT-1 was digested with Bam HI and BglII and thenreligated. In each case, the 5' part of the LAV genome was retained withthe vector. HB101 cells were transformed with the ligated DNA andcolonies containing the pSS-5 and pBS-5 inserts were identified byrestriction analysis of the purified plasmid DNA.

Regions of pSS-5 and pBS-5 were then subcloned into the intermediatevector, pIC19R. As discussed above, this provided the necessaryrestriction sites in the correct reading frame for transfer to theexpression vectors.

Specifically, the Hind III fragment of pBS-5 (bp 631 to bp 1258 of theLAV genome; numbering according to Wain-Hobson et al., Cell 40: 9(1985)) was ligated into Hind III and calf intestinal phosphatasetreated pIC19R (FIG. 4). The ligated DNA was taken up by CaCl₂ -shockedE. coli TB-1. Using the chromagenic substrate5-bromo-4-chloro-3-indodlyl-βgalactosidase (Sigma), ampicillin resistantcolonies were screened for inactivation of β-galactosidase due toinsertion of the gag sequence. The orientation of the insert wasdetermined by digesting plasmid DNA with Pvu II. The resultant plasmidis referred to as pSM002.

The Pvu II-Bgl II fragment (bp 691 to bp 1642 of the LAV genome) wasligated into Sma I and Bgl II digested pIC19R (FIG. 5). The ligated DNAwas taken up by E. coli HB101 and the resultant ampicillin resistantcolonies screened with the chromogen as described above. Candidatecolonies were further screened by restriction analysis of plasmid DNA.The resultant plasmid is referred to as pBPB14.

2. Insertion of the gag sequence into trp vectors

The expression vectors contained the E. coli trp operon promotor,operator, and trp E gene inserted into pBR322 (FIG. 2). The trp E genewas truncated at its 5'-most Bgl II site by the insertion of apolylinker sequence (Konopka et al., J. Virol. 51: 223 (1984)). Thedifferent trp vectors (pJH 12 and pJH 14) differed according to thereading frame of the restriction sites within the polylinker region.Insertion of an open reading frame into the appropriate vector resultsin the production of a fusion protein with trp E sequences at the aminoterminal end (Spindler et al., J. Virol. 49: 132 (1984)).

pGAG-2 (ATCC Accession #53111) was constructed by digesting the Hind IIIgag subclone (pSM002, FIG. 4) with Bgl II and Bam Hl. Bgl II and Bam Hlsites were located within the bracketing polylinker region of pIC19R.The gag fragment was gel purified and ligated into Bam Hl digested pJH12(FIG. 4). Ligated DNA was taken up by CaCl₂ -shocked E. coli HB101 andcolonies were grown in the presence of ampicillin (100 ug/ml) andtryptophan (40 ug/ml). Tryptophan was used to suppress expression of theforeign protein, accumulation of which may be deleterious to thebacteria. Candidate ampicillin resistant colonies were identified byhybridization with a radioactively labeled gag DNA probe. Restrictionanalysis of minilysates was used to confirm the presence and orientationof the insert.

pGAG-3 (ATCC Accession #53112) was constructed by EcoRl digestion ofpBPB14, which contains the gag region between Pvu II (bp 691) and Bgl II(bp 1642), as shown in FIG. 5. The EcoRl sites are in the polylinkersequences bracketing the gag sequences. The gag fragment was gelpurified and ligated into EcoRl and calf intestinal phosphatase treatedpJH14. E. coli HB101 cells were transformed with the ligated DNA, platedas described above, and screened by restriction analysis to confirm thepresence and orientation of the gag sequence within pJH14.

Escherichia coli transformants containings pGAG-2 have been deposited asATCC Deposit No. 53111 and Escherichia coli transformants containingpGAG-3 have been deposited as ATCC Deposit No. 53112 at The AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 onMay 2, 1985.

B. Protein expression

1. Transformation of E. coli with the trp-gag constructs

Each of the recombinant trp-gag expression plasmids was transferred fromE. coli HB101 into E. coli C600, because the latter is a better host forprotein production. Transfer involved transformation of CaCl₂ -shockedC600 with supercoiled DNA from minilysates of HB101. Bacteria wereplated in the presence of ampicillin and tryptophan as described(Konopka et al., J. Virol. 51: 223 (1984)). Drug-resistant colonies werescreened by minilysates to confirm the presence of the appropriateplasmid.

2. Expression of trp-gag proteins

Growth and induction of E. coli C600 transformed by the trp expressionvectors were as described (Spindler et al., J. Virol. 49: 132 (1984);Konopka et al., J. Virol. 51: 223 (1984)). Briefly, minimal mediumcontaining tryptophan (40 ug/ml) and ampicillin (100 ug/ml) wasinoculated with transformed bacteria from glycerol stocks. Cultures weregrown with aeration at 37° C. overnight. The overnight cultures werethen inoculated at 1:100 into fresh minimal medium containing ampicillin(100 ug/ml) but no tryptophan. These cultures were grown with aerationfor 2-3 hours (up to early log phase) at 37° C. The inducer,3-β-indoleacrylic acid (Sigma), was added to a final concentration of 20ug/ml from freshly made stocks at 20 mg/ml in 95% ethanol.

Induced cultures were grown at 37° C with aeration for 4 to 5 hours andthen pelleted and, optionally, frozen. Protein yields from pGAG-2 andpGAG-3 were typically between 10-30 mg/liter.

C. Isolation and purification of trp-gag proteins

Fusion proteins were partially purified from cell pellets as described(Konopka et al., J. Virol. 51: 223 (1984)). Briefly, bacteria wereresuspended in 100 ml of 50 mM Tris, pH 7.5/0.5 mM EDTA/150 mM NaCl(TNE) per liter of induced culture. Lysozyme (Sigma) was added to afinal concentration of 1 mg/ml. After fifteen minutes at 0° C, NP40 wasadded to the mixture to a final concentration of between 0.05% and 0.2%for ten minutes at 0° C. 1-2 mg of DNase (Sigma) was then added with 150ml of DNase buffer (1.5 M NaCl/12 mM MgCl₂). Reaction mixtures wereincubated until they were no longer viscous, usually several hours toovernight. Insoluble proteins were then pelleted by centrifugation for15 minutes at 8000 xg at 0° C. Pellets were washed two times in TNE andthen analyzed for the presence of insoluble proteins by denaturingpolyacrylamide gel electrophoresis. Proteins were visualized by stainingwith Coomassie brilliant blue.

Alternatively, the insoluble pellet was denatured and reduced byresuspending the pellet from about 200 ml of cells in 180 ul of 0.14 MTris HCl, pH 7.8/6% SDS and 20 ul of β-mercaptoethanol and heating at95° C.-100° C. for 20 minutes. The sample was then dried in a Speed VacConcentrator (Savant Instruments, Hicksville, NY). In order to removethe SDS, the sample was extracted as follows: the pellet was resuspendedin 1 ml of acetone/triethylamine/acetic acid/water (17/1/1/1) andvigorously vortexed, the suspension was chilled on ice for one hour,centrifuged and the supernatant discarded. This extraction was repeatedtwice with 0.4 ml of the above acetone mixture, and then twice with 0.4ml of acetone alone. The pellet was then dried in the Speed VacConcentrator.

The protein pellet was then dissolved in 1.4 ml of 6M guanidinehydrochloride/0.1 M Tris HCl, pH 8.5/10 mM DTT and incubated at 37° C.for 4 hours. Any particulate material was removed by centrifugation. Thesample, contained in the supernatant, was loaded onto a 1.6×96 cmSephacryl S-300 column (Pharmacia, Piscataway, N.J.) equilibrated in 6 Mguanidine hydrochloride/10 mM EDTA/1 mM DTT. The column was eluted withthe same buffer and the absorbance of the eluted material was measuredat 280 nm.

The fractions were assayed by ELISA with LAV seropositive sera and withE. coli seropositive sera, to determine specific and contaminatingreactivities, respectively. The fractions showing reactivity with theformer but not with the latter serum were used in subsequent ELISA ofpatient sera.

D. Immunological reactivity of trp-gag proteins

1. Analysis by Western blots

Aliquots from the insoluble protein preparations expressed by pGAG-2 andpGAG-3 were solubilized in 2% sodium dodecylsulfate/100 mM Tris, pH6.8/20% glycerol/1.5 M β-mercaptoethanol and electrophoresed ondenaturing polyacrylamide gels. Proteins were electrotransferred ontonitrocellulose (BA85, Schleicher and Schuell, Keene, N.H.) and thefilters blocked with 5% bovine serum albumin (Sigma). Filters were thenprobed with E. coli-adsorbed human sera pooled from AIDS patients. Thefilters were developed with HRP-conjugated goat α HuIg. The pool wasreactive with both trp-gag fusion proteins but not with trp E proteinalone.

2. Analysis by ELISA

Column-purified GAG-2 and GAG-3 proteins were diluted in 0.05 Mcarbonate/bicarbonate buffer (pH 9.6) to a final concentration of 0.3ug/ml and 3.4 ug/ml, respectively. Fifty ul aliquots were loaded permicrotiter well and incubated at 4° C. overnight. Plates were thenblocked with BLOTTO (5% [w/v] nonfat dry milk/0.01% thimerosol/0.01%antifoam A in 0.01 M sodium phosphate, pH 7.2/0.15 M sodium chloride)for one hour at room temperature. Sera were diluted 1:100 with a 1:1mixture of BLOTTO and PBS (0.01 M sodium phosphate, pH 7.3/0.15 M NaCl),and 50 ul of diluted sera was added per well for one hour at 37° C. Thesera were removed, and the plates were washed three times in wash buffer(0.15 M NaCl/0.05% [w/v]Tween 20) before adding 100 ul of the goatanti-human IgG/horseradish peroxidase conjugate (50% stock diluted1:10,000 in 50 mM NaCitrate/0.05% Tween 20/1% heat-inactivated normalgoat serum; obtained from Antibodies, Inc., Davis, Calif.) for one hourat 37° C. The conjugate was removed and the plates washed three timeswith 0.15 M NaCl/0.05% (w/v) Tween 20. The ELISA was developed by adding100 ul/well of substrate solution (10 mg 3,3', 5,5'-tetramethylbenzidinein 50 ml 0.05 M sodium citrate, pH 7.0) for 30 minutes at roomtemperature. Reactions were stopped with 100 ul/well of 3N H₂ SO₄, andthe optical density at 450 nm determined by an automated ELISA reader.Proteins produced by pGAG-2 and pGAG-3 were both found to be reactivewith a panel of known seropositive sera.

The panel included sera from two healthy heterosexuals, five individualsdiagnosed as LAS (lymphadenopathy syndrome) and/or homosexual, oneindividual with AIDS, and a pool of sera from AIDS patients. The twoheterosexual sera scored negative in a whole virus ELISA. All sera fromAIDS, LAS and/or homosexual individuals were confirmed as seropositivein a whole virus ELISA and by radiolabeled immunoprecipitation of LAVantigens. The results from these sera are shown in Table I. Thesefindings demonstrate that sera reactive to LAV antigens are alsoreactive to our bacterially expressed gag proteins.

3. Fluorescence slide test for detection of serum antibody to LAV

Soluble protein produced as described above is conjugated to latexbeads, and the protein/bead preparation is ethanol fixed onto microscopeslides. An aliquot of patient serum is incubated with the protein/beadson a slide. The slides are washed, and FITC-labeled anti-humanimmunoglobulin in Evans blue counterstain is added. The slides arewashed, and mounting medium and coverslip applied to each.

Alternatively, the protein/bead preparation is placed in test tubes forincubation with patient serum. The tubes are centrifuged and washed, andthe FITC-labeled anti-human immunoglobulin in Evans blue counterstain isadded. The tubes are centrifuged and the supernatant aspirated. Analiquot of the beads is placed on a microscope slide and ethanol fixed,and coverslips are mounted.

All slides are examined by fluorescence microscopy. If test serum isantibody positive, beads appear as fluorescent green spheres; if testserum is antibody negative, beads appear as red spheres.

                  TABLE I                                                         ______________________________________                                        Comparison of GAG-2 and GAG-3 with a Whole Virus Lysate in                    an ELISA for the Detection of Antibodies to LAV                                                Whole                 Confirmed                              Serum            Virus                 as                                     No.   Diagnosis  Lysate  GAG-2  GAG-3  Seropos.                               ______________________________________                                        Y1/   positive   2.000   1.292  1.580  yes                                    CDC   control pool                                                            124   LAS and/or 1.189   0.762  0.396  yes                                          homosexual                                                              127   LAS and/or 1.046   0.291  1.093  yes                                          homosexual                                                              130   LAS and/or 0.912   0.420  1.200  yes                                          homosexual                                                              153   LAS and/or 2.000   1.137  1.073  yes                                          homosexual                                                              501   AIDS       1.109   1.049  1.556  yes                                    666   unknown    2.000   n.d.   1.747  yes                                    637   healthy    0.097   0.113  0.116  not sero-                                    heterosexual                     positive                               641   healthy    0.199   0.116  0.069  not sero-                                    heterosexual                     positive                               ______________________________________                                    

4. Reactivity of combination trp-gag and trp-env proteins

A trp-gag protein was combined with a trp-env protein in a microtiterwell. The ELISA was then performed as describe above for GAG-2 or GAG-3alone. Table II shows that the combination of GAG-3 and ENV-3 has ahigher sensitivity for detecting seropositive individuals than foreither protein alone. Of the seropositive samples, 7/7 were detectedwhen the proteins were combined, whereas 6/7 were detected with GAG-3 orENV-3 alone.

                                      TABLE II                                    __________________________________________________________________________    Comparison of Combined GAG-3 and ENV-3 with GAG-3 or ENV-3                    Alone or for the Detection of Antibodies to LAV                                              GAG-3                                                                     Whole                                                                             100 ng                                                                            0   100 ng                                                                            50 ng                                                                             Confirmed                                      Serum      Virus                                                                             ENV-3           as LAV                                         No. Diagnosis                                                                            Lysate                                                                            0   100 ng                                                                             70 ng                                                                            70 ng                                                                             Seropos.                                       __________________________________________________________________________     591                                                                              AIDS   1.109                                                                             1.663                                                                             2.039                                                                             2.271                                                                             1.696                                                                             yes                                              7 LAS and/or                                                                           2.0 0.394                                                                             0.498                                                                             0.742                                                                             0.627                                                                             yes                                                homosexual                                                                 153                                                                              LAS and/or                                                                           2.0 0.542                                                                             2.192                                                                             2.178                                                                             2.167                                                                             yes                                                homosexual                                                                 154                                                                              LAS and/or                                                                           1.069                                                                             0.497                                                                             2.054                                                                             2.167                                                                             2.007                                                                             yes                                                homosexual                                                                1296                                                                              blood donor                                                                          2.000                                                                             0.278                                                                             0.383                                                                             0.545                                                                             0.471                                                                             yes                                            1642                                                                              LAS and/or                                                                           0.998                                                                             0.109                                                                             1.529                                                                             1.776                                                                             1.768                                                                             n.d.                                               homosexual                                                                1709                                                                              healthy                                                                              0.133                                                                             0.071                                                                             0.164                                                                             0.152                                                                             0.155                                                                             n.d.                                               heterosexual                                                              1890                                                                              healthy                                                                              n.d.                                                                              0.214                                                                             0.175                                                                             0.286                                                                             0.269                                                                             not                                                heterosexual               sero-                                                                         pos.                                           1891                                                                              healthy                                                                              n.d.                                                                              0.154                                                                             0.155                                                                             0.299                                                                             0.279                                                                             not                                                heterosexual               sero-                                                                         pos.                                            923                                                                              blood donor                                                                          0.119                                                                             0.178                                                                             0.476                                                                             0.337                                                                             0.325                                                                             n.d.                                           __________________________________________________________________________     n.d. = Not determined.                                                   

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notto be limited except as by the appended claims.

We claim:
 1. An isolated and purified DNA sequence comprising a portionof the gag region of the LAV genome, said portion corresponding to thatfrom bp631 to bp1258 or from bp691 to bp1642 of LAV and coding for aprotein which comprises a fragment of at least one virally encoded gagprotein and which protein fragment is immunologically reactive withantibodies to LAV.
 2. The isolated and purified DNA sequence of claim 1wherein said sequence is pGAG-2.
 3. The isolated and purified DNAsequence of claim 1 wherein said sequence is pGAG-3.
 4. An isolated andpurified DNA sequence comprising a portion of the gag region of the LAVgenome, wherein said portion corresponds to bp631 to bp1258, saidportion coding for a fragment of a gag protein which is immunologicallyreactive with antibodies to LAV.
 5. An isolated and purified DNAsequence comprising a portion of the gag region of the LAV genome,wherein said portion corresponds to bp691 to bp1642, said portion codingfor a fragment of a gag protein which is immunologically reactive withantibodies to LAV.
 6. A recombinant plasmid capable of stablereplication and expression in bacterial host cells, said plasmidincluding procaryotic transcriptional and translational signal for saidexpression, followed in reading phase by a DNA sequence comprising aportion of the gag region of the LAV genome corresponding to bp631 tobp1258 or bp691 to bp1642, said portion coding for a protein whichcomprises a fragment of at least one virally encoded gag protein andwhich protein fragment is immunologically reactive with antibodies toLAV.
 7. The recombinant plasmid of claim 6 wherein said expression isinducible.
 8. The recombinant plasmid of claim 7 wherein said signalsare derived from the trp operon.
 9. A bacterial cell stably transformedwith a recombinant plasmid capable of replication and expression inbacterial host cells, said plasmid including procaryotic transcriptionaland translational signals for said expression, followed in reading phaseby a DNA sequence comprising a portion of the gag region of the LAVgenome corresponding to bp631 to bp1258 or bp691 to bp1642 and codingfor a protein which comprises a fragment of at least one virally encodedgag protein and which protein fragment is immunologically reactive withantibodies to LAV.
 10. The transformed cell of claim 9 wherein saidbacterial cell is E. coli.
 11. The transformed cell of claim 9 whereinsaid expression is inducible.
 12. The transformed cell of claim 11wherein said signals are derived from the trp operon.
 13. A method forpreparing proteins which are immunologically reactive with antibodies toLAV, comprising:introducing into a bacterial host cell a recombinantplasmid capable of stable replication and expression in bacterial hostcells, said plasmid including procaryotic transcriptional andtranslational signals for said expression, followed in reading phase bya DNA sequence comprising a portion of the gag region of the LAV genomewhich corresponds to bp631 to bp1258 or to bp691 to bp1642, said portioncoding for a protein which comprises a fragment of at least one virallyencoded gag protein and which protein fragment is immunologicallyreactive with antibodies to LAV; growing said bacterial host in anappropriate medium for said expression; and isolating the proteinproduct of said sequence from said bacterial host.
 14. The method ofclaim 13, including, after isolation of said protein product, purifyingsaid product by gel permeation chromatography.
 15. The method of claim13 wherein the expression of said protein is induced by3-β-indoleacrylic acid.
 16. The method of claim 13 wherein saidexpression is inducible.
 17. The method of claim 16 wherein said signalsare derived from the trp operon.
 18. A method for preparing proteinswhich are immunologically reactive with antibodies to LAV,comprising:introducing into a procaryotic cell a recombinant vectorcapable of stable replication and expression in said procaryotic cells,said vector including transcriptional and translational signals for saidexpression, followed in reading phase by a DNA sequence comprising aportion of the gag region of the LAV genome, which portion correspondsto bp631 to bp1258 and codes for a fragment of a gag protein which isimmunologically reactive with antibodies to LAV; growing saidprocaryotic cell in an appropriate medium for said expression; andisolating the protein product of said sequence from said procaryoticcell.
 19. The method of claim 18, including, after isolation of saidprotein product, purifying said product by gel permeationchromatography.
 20. The method of claim 18, wherein the procaryotic cellis bacterial and the recombinant vector is a plasmid.
 21. The method ofclaim 20, wherein said expression is inducible and/or suppressible. 22.A method for preparing proteins which are immunologically reactive withantibodies to LAV, comprising:introducing into a procaryotic cell arecombinant vector capable of stable replication and expression in saidprocaryotic cells, said vector including transcriptional andtranslational signals for said expression, followed in reading phase bya DNA sequence comprising a portion of the gag region of the LAV genome,which portion corresponds to bp691 to bp1642 and codes for a fragment ofa gag protein which is immunologically reactive with antibodies to LAV;growing said procaryotic cell in an appropriate medium for saidexpression; and isolating the protein product of said sequence from saidprocaryotic cell.
 23. The method of claim 22, including, after isolationof said protein product, purifying said product by gel permeationchromatography.
 24. The method of claim 22, wherein the procaryotic cellis bacterial and the recombinant vector is a plasmid.
 25. The method ofclaim 24, wherein said expression is inducible and/or suppressible.